Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method

Wei, Yanyan; Zhan, Qun-Mei; Zhu, Xiao-feng; Yan, Aifen; Feng, Juan; Li, Jian‐Hao; Tang, Dongsheng

doi:10.1007/s10529-020-02930-0

Cited by 9 publications

(14 citation statements)

References 34 publications

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“…Gene sequencing showed that 33.3% (4/12) of single-cell colonies had biallelic ABE-mediated Ato-G conversions at the target locus. This e ciency is comparable to that of CRISPR/Cas9 in generating geneknockout pig cells conducted in our previous studies, suggesting that the ABE system is a practical and feasible approach to generating genetically modi ed porcine cell colonies (Zhu et al 2018;Wei et al 2020;Pan et al 2021). More importantly, RT-PCR and Western blotting tests showed that ABE-mediated single-base mutations in the conservative splice acceptor in the intron 5 of GHR successfully induced exon 6 skipping at the RNA level and gene knockout at the protein level.…”

Section: Discussionsupporting

confidence: 71%

“…Procedures used for the isolation, cultivation, and transfection of kidney broblasts from newborn Bama minipigs were based upon our previous studies (Zhu et al 2018;Wei et al 2020;Pan et al 2021). After deep anesthetization, the newborn piglet was euthanized and both kidneys were removed and minced in Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA).…”

Section: Preparation Of Bama Minipig Broblast Cellsmentioning

confidence: 99%

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Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Zhu

Pan

Lin

et al. 2021

Preprint

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Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A·T pairs to G·C pairs in the genome without generating DNA double-strand breaks, and this method has higher accuracy and biosafety in pig genetic modification. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the base-editing window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine base-editing-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We first constructed a modified “all-in-one” ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The “all-in-one” ABE vector induced efficient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3’ end of the intron 5) and splice donor site (GT sequence at the 5’ end of the intron 6) in the porcine gene GHR; this method achieved highly efficient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the first proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modification of pigs for agricultural and medical applications.

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Section: Discussionsupporting

confidence: 71%

Section: Preparation Of Bama Minipig Broblast Cellsmentioning

confidence: 99%

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Zhu

Pan

Lin

et al. 2021

Preprint

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“…Design and construction of the CBE plasmid were performed according to our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). The sgRNAs used in this study were designed as described below, and were produced by BGI company.…”

Section: Construction Of Cbe Plasmid For Transfectionmentioning

confidence: 99%

“…Procedures used for the isolation, cultivation, and transfection of kidney broblasts from newborn Bama minipigs were based upon our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). After deep anesthetization, the newborn piglet was euthanized and both kidneys were removed and minced in Dulbecco's phosphate-buffered saline (DPBS; Gibco, Grand Island, NY, USA).…”

Section: Preparation Of Bama Minipig Broblast Cellsmentioning

confidence: 99%

“…Potential OTSs were predicted by the CRISPR Design Tool (http://crispor.tefor.net) according to our previous studies (Wei et al, 2020;Zhu et al, 2020a;b). Six sites with potential genomic off-target effects for MSTN locus were selected for detection of off-targets occurred in base-edited cell colonies (Table 3).…”

Section: Genomic Off-target Detection In Base-edited Cell Coloniesmentioning

confidence: 99%

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Application of the modified cytosine base-editing in the cultured cells of bama minipig

et al. 2021

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Bama minipig is a unique miniature swine bred from China. Their favorable characteristics include delicious meat, strong adaptability, tolerance to rough feed, and high levels of stress tolerance. Unfavorable characteristics are their low lean meat percentage, high fat content, slow growth rate, and low feed conversion ratio. Genome-editing technology using CRISPR/Cas9 e ciently knocked out the myostatin gene (MSTN) that has a negative regulatory effect on muscle production, effectively promoting pig muscle growth and increasing lean meat percentage of the pigs. However, CRISPR/Cas9 genome editing technology is based on random mutations implemented by DNA double-strand breaks, which may trigger genomic off-target effects and chromosomal rearrangements. The application of CRISPR/Cas9 to improve economic traits in pigs has raised biosafety concerns. Base editor (BE) developed based on CRISPR/Cas9 such as cytosine base editor (CBE) effectively achieve targeted modi cation of a single base without relying on DNA double-strand breaks. Hence, the method has greater safety in the genetic improvement of pigs. The aim of the present study is to utilize a modi ed CBE to generate MSTN-knockout cells of Bama minipigs. Our results showed that the constructed "all-in-one"-modi ed CBE plasmid achieved directional conversion of a single C•G base pair to a T•A base pair of the MSTN target in Bama miniature pig broblast cells. We successfully constructed multiple single-cell colonies of Bama minipigs broblast cells carrying the MSTN premature termination and veri ed that there were no genomic off-target effects detected. This study provides a foundation for further application of somatic cell cloning to construct MSTN-edited Bama minipigs that carry only a single-base mutation and avoids biosafety risks to a large extent, thereby providing experience and a reference for the base editing of other genetic loci in Bama minipigs.

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Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells

et al. 2022

Self Cite

View full text Add to dashboard Cite

Gene-knockout pigs have important applications in agriculture and medicine. Compared with CRISPR/Cas9, Adenine base editor (ABE) convert single A•T pairs to G•C pairs in the genome without generating DNA doublestrand breaks, and this method has higher accuracy and biosafety in pig genetic modi cation. However, the application of ABE in pig gene knockout is limited by protospacer-adjacent motif (PAM) sequences and the baseediting window. Alternative mRNA splicing is an important mechanism underlying the formation of proteins with diverse functions in eukaryotes. Spliceosome recognizes the conservative sequences of splice donors and acceptors in a precursor mRNA. Mutations in these conservative sequences induce exon skipping, leading to proteins with novel functions or to gene inactivation due to frameshift mutations. In this study, adenine baseediting-mediated exon skipping was used to expand the application of ABE in the generation of gene knockout pigs. We rst constructed a modi ed "all-in-one" ABE vector suitable for porcine somatic cell transfection that contained an ABE for single-base editing and an sgRNA expression cassette. The "all-in-one" ABE vector induced e cient sgRNA-dependent A-to-G conversions in porcine cells during single base-editing of multiple endogenous gene loci. Subsequently, an ABE system was designed for single adenine editing of the conservative splice acceptor site (AG sequence at the 3' end of the intron 5) and splice donor site (GT sequence at the 5' end of the intron 6) in the porcine gene GHR; this method achieved highly e cient A-to-G conversion at the cellular level. Then, porcine single-cell colonies carrying a biallelic A-to-G conversion in the splice acceptor site in the intron 5 of GHR were generated. RT-PCR indicated exon 6 skipped at the mRNA level. Western blotting revealed GHR protein loss, and gene sequencing showed no sgRNA-dependent off-target effects. These results demonstrate accurate adenine base-editing-mediated exon skipping and gene knockout in porcine cells. This is the rst proof-of-concept study of adenine base-editing-mediated exon skipping for gene regulation in pigs, and this work provides a new strategy for accurate and safe genetic modi cation of pigs for agricultural and medical applications. Key Points1. ABE can triggers e cient single base conversion at multiple gene loci in pig cells.2. ABE-mediated exon skipping provides an alternative strategy for gene KO in pig cells.

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Efficient CRISPR/Cas9-mediated gene editing in Guangdong small-ear spotted pig cells using an optimized electrotransfection method

Cited by 9 publications

References 34 publications

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Adenine Base-Editing-Mediated Exon Skipping Induces Gene Knockout in Cultured Pig Cells

Application of the modified cytosine base-editing in the cultured cells of bama minipig

Adenine base-editing-mediated exon skipping induces gene knockout in cultured pig cells

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